Diagnosis and monitoring of colon cancer patients by measurement of NCA 50/90 in blood

ABSTRACT

A method for aiding in the diagnosis of, and monitoring the progression or course of, colon cancer in a patient by measuring the amount of NCA 50/90 in a blood sample, e.g., serum sample, obtained from the patient. Measurement in a single sample of an amount of NCA 50/90 significantly higher than the mean amount of NCA 50/90 in the normal population is an indication of colon cancer in a symptomatic patient. The course of colon cancer can also be monitored by performing a series of specific immunoassays over time to determine changes in the level of NCA 50/90 in blood samples. Increases in blood NCA 50/90 levels over time are indicative of a deteriorating condition whereas decreasing levels of blood NCA 50/90 over time indicate an improving condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application under 37 CFR § 1.60 of pending priorapplication Ser. No. 08/400,213, filed Mar. 7, 1995, now abandoned,which is a continuation of abandoned application Ser. No. 08/193,975,filed Feb. 9, 1994 now abandoned, which is a continuation-in-part ofabandoned application Ser. No. 08/050,935, filed Apr. 21, 1993 nowabandoned.

BACKGROUND OF THE INVENTION

The present invention relates to the diagnosis of, and monitoring theprogression, course, or stage of, disease in colon cancer patients. Moreparticularly, the invention relates to such diagnostic and monitoringmethods based on measurement of cancer marker blood levels.

A number of substances have been determined to be useful markers inmonitoring the course of various cancer types. Some useful markers thathave been identified are oncofetal antigens such as carcinoembryonicantigen (CEA) and alpha-fetoprotein, tissue-specific antigens such asprostate-specific antigen (PSA), and mucin antigens such as thoseconventionally known as CA-125 and CA-19-9. Immunoassays for antigenssuch as these are typically used as confirmatory tests at the time ofdiagnosis and subsequently for monitoring patient status. Occasionally,the use of such tests crosses the boundaries of tumor type (for example,the use of CEA tests in colon, breast, and lung cancer, andalpha-fetoprotein in hepatocellular and testicular cancer), but theutility of each test type is foremost for a single tumor type (forexample, PSA for prostate cancer and CA-125 for ovarian cancer)

A family of antigenic proteins have been identified which aregenetically and immunologically related to CEA (Thompson, J. and W.Zimmerman (1988) Tumor Biol. 9, 63-83; and Barnett, T. and W. Zimmerman(1990) Tumor Biol. 11, 59-63). Among these are the nonspecificcross-reacting antigens (NCAs), the trans-membrane antigens designatedbiliary glycoprotein (BGP, and sometimes referred to as TM-CEAs), andthe family of pregnancy-specific β-glycoproteins (PSGs) (for adescription of the accepted nomenclature of these genes and theirprotein products, reference can be made to: Barnett, T. and W. Zimmerman(1990) Tumor Biol. 11, 59-63). Molecular cloning of the CEA gene familyhas enabled the identification of 22 members, of which 20 are probablyexpressed (Frangsmyr, L. et al. (1992) Tumor Biol. 13, 98-99; andHammerstrom, S. et al Tumor Biol. 13, 57). The results of moleculargenetic analysis have given a better understanding of the complex groupof glycoproteins in the CEA gene family.

NCA was originally described as a component of normal tissue whichcross-reacted with antibodies raised to CEA (Mach, J.-P. and G.Pusztaszeri (1972) Immunochemistry 9, 1031-1034; and von Kleist, S.,Chavenel, G. and P. Burtin (1972) Proc. Natl. Acad. Sci. USA 69,2492-2494). As such, NCA was considered a potential non-tumor derivedinterferant in assays for CEA. Molecular cloning identified one speciesof NCA of calculated M_(r) 37,000 designated by one group as NCA-BT(Barnett, T., Goebel, S. J., Nothdurft, M. A. and J. J. Elting (1988)Genomics 3, 59-66) to denote the breast tissue origin of the clonedcDNA, and by others as NCA (Tawargi, Y. et al. (1988) Biochem. Biophys.Res. Commun. 150, 89-96; and Neumaier, M. et al (1988) J. Biol. Chem.263, 3203-3207). This single NCA species has since been termed NCA 50/90(Kolbinger, F., Schwarz, K., Brombacher, F., von Kleist, S., andGrunert, F. (1989) Biochem. Biophys. Res. Commun. 161, 1126-1134)because it was now known to be processed into two mature isoforms ofM_(r) 50,000 and 90,000 which have different degrees of glycosylation. Asecond and distinct NCA gene was subsequently identified by molecularcloning from leukemic cells that codes for an M_(r) 95,000 glycoprotein(Kuroki, M. et al (1991) J. Biol. Chem. 266, 11810-11817). This latterNCA has been termed NCA 95.

Early studies also identified a cross-reacting antigen from adult stoolsand from meconium which, for historical reasons, was termed NCA-2(Burtin, P., Chavenel, G. and H. Hirsch-Marie (1973) J. Immunol. 111,1926-1928). Indeed, a recent report suggests that variability in CEAresults obtained with different commercial kits may be due tointerference with NCA-2 (O. P. Bormer (1991) Clin. Chem. 37, 1736-1739).The designation of this antigen as NCA is, however, a misnomer. It hasbeen identified as a proteolytic fragment of CEA, since the first 30amino acids of the meconium-derived NCA-2 are identical in sequence withCEA (Siepen, D. et al (1987) Biochem. Biophys. Res. Commun. 174,212-218). In contrast, cDNAs for NCA 50/90 have been described and codefor distinct and different amino acid sequences in this region.

Given the improved understanding of the CEA gene family resulting frommolecular cloning analysis, monoclonal antibodies can now be identifiedwhich recognize specific family members and do not cross react withclosely related molecules. Previous attempts to raise antibodies to NCAhave been plagued with the problem of cross reactivity with CEA familymembers. This may explain why NCA has been considered a poor serummarker for cancer diagnosis and monitoring (Shively, J. E., Spayth, V.,Chang, F.-F., Metter, G. E., Klein, L., Present, C. A., and C. W. Todd(1982) Cancer Res. 42, 2502-2513; and Burtin, P., Chavenel, G.,Hendrick, J. C. and N. Frenoy (1986) J. Immunol. 137, 839-845). It hasbeen further speculated that NCA-specific monoclonal antibodies such asare now widely accepted for CEA and other antigens would be verydifficult to develop (Burtin, P. et al., supra).

In addition, it is now clear that members of the CEA gene family aredifferentially expressed by various tumor types. For example, it is wellknown that CEA is expressed in most if not all colorectal carcinomas,while expression is limited to a minority of breast carcinomas. Prior tothe generation of specific monoclonal antibodies, attempts to quantitateNCA levels in the serum of cancer patients were confounded by thepresence of other CEA gene family members that cross reacted with theantibodies being used. However, because of the successful production ofmonoclonal antibodies specific to NCA 50/90, it is now possible todetermine the incidence of elevated NCA 50/90 protein in differentcancer types.

Although there have been reports of monoclonal antibodies specific forNCA 50/90 (Chavenel, G., Frenoy, N., Escribano, M. J. and P. Burtin(1983) Oncodev. Biol. and Med. 4, 209-217; and Yeung, M., M.-W.Hamrnmerstrom, M. L., Baranov, V. and S. Hammerstrom (1992) Tumor Biol.9, 119), there have been no reports of a monoclonal antibody which bindsto NCA 50/90 but does not recognize any other CEA family membersincluding CEA, NCA 95, NCA 2, BGP and PSG. Similarly, several reportshave suggested that NCA may be elevated in the serum of cancer patientswith solid tumors (von Kleist, S., Troupel, S., King, M. and P. Burtin(1977) Br. J. Cancer 35, 875-880; and Wahren, B., Gahrton, G., Ruden, U.and S. Hammerstrom (1982) Int. J. Cancer 29, 133-137; and Harlozinska,A., Rachel, F., Gawlikowski, W., Richter, R. and J. Kolodziej (1991)Eur. J. Surg. Oncol. 17, 59-64; and Reck, W., Daniel, S., Nagel, G.,Him, M., von Kleist, S., and F. Grunert (1992) Tumor Biol. 13, 110-111),but these measurements used antibodies that have not been shown torecognize NCA 50/90 to the exclusion of other CEA-related molecules. Inaddition, there have been no reports of a correlation between blood NCAlevels and the clinical status of patients with solid tumors.

Results of several studies have shown that NCA is elevated in the serumof patients with leukemia, particularly chronic myelocytic leukemia(Frenoy, F. and P. Burtin (1980) Clin. Chim. Acta 103, 23-31; Wahren,B., Gahrton, G. and S. Hammarstrom (1980) Cancer Res. 40, 2039-2044;Wahren, B., Gahrton, G. and S. Hammarstrom (1980) Cancer Res. 40,2039-2044; Wahren, B., Gahrton, G., Ruden, U. and S. Hammarstrom (1982)Int. J. Cancer 29, 133-137; Frenoy, N., Ben-Bunant, M., Burreul, C.,Child, J. A., Gendron, M. C., Missett, J. L., Razafimahaleo, E. and P.Burtin (1982) Br. J. Cancer 46, 765-772). These data show that NCA maybe elevated in the early stages of leukemia, but that serum NCA levelsdecrease during blast crisis. It is not clear from these studies ifchanges in NCA levels reflect changes in the clinical status of thepatients since, in one study, changes in the serum concentration of NCAwere reflected by changes in total white cell counts andpolymorphonuclear cell counts (Wahren, B. Gahrton, G., Ruden, U. and S.Hammarstrom (1982) Int. J. Cancer 29, 133-137), whereas in otherstudies, these same parameters either did not correlate or correlatedonly poorly (Frenoy, F. and P. Burtin (1980) Clin. Chim. Acta 103,23-31; Frenoy, N., Ben-Bunant, M., Burruel, C., Child, J. A., Gendron,M. C., Missette, J. L., Razafimahaleo, E. and P. Burtin (1982) Br. J.Cancer 46, 765-772). In any event, these studies are difficult tointerpret since the methods used to measure NCA in serum used polyclonalantibodies which were not well characterized with respect to theirreactivity with various members of the CEA family of molecules.

Previous attempts to quantitate the level of NCA 50/90 in the serum havebeen hampered by the lack of a suitable standard. Measurements of NCA inblood have shown mean values in serum from normal individuals of from 30ng/ml (Harlozinska, A., et al. supra) to 130 ng/ml (von Kleist, S.,Troupel, S., King, M. and P. Burtin (1977) Br. J. Cancer 35, 875-880).This is due to the use of biochemically purified NCA as a standard tocalibrate immunoassay measurements of NCA in blood and blood fluids.Just as the monoclonal antibodies have not been demonstrated tospecifically recognize NCA 50/90, neither has the purity of the NCAstandard preparations been determined.

U.S. patent application Ser. No. 815,934, filed Dec. 30, 1991, andentitled “Monitoring of NCA-BT in Blood Samples of Breast CancerPatients”, reports the finding that changes in the blood level of NCA50/90 (therein designated as NCA-BT) in breast cancer patients providesa means for monitoring the progression of the disease. In particular, itwas found that increases in blood NCA 50/90 levels measured byperforming a series of specific immunoassays over time indicated adeteriorating condition in a significant number of patients, whiledecreases in blood NCA 50/90 levels indicated an improving condition insuch patients.

SUMMARY OF THE INVENTION

It has now been found that NCA 50/90 can be significantly elevated inthe blood of patients with colon cancer. Accordingly, the presentinvention provides a method for aiding in the diagnosis of colon cancerin a patient who presents with symptoms of colon cancer (i.e., asymptomatic patient), comprising the steps of determining the amount ofNCA 50/90 in a blood sample obtained from said patient and comparingsuch measured amount of NCA 50/90 to the mean amount of NCA 50/90 in thenormal population, whereby the presence of a significantly increasedhigher amount of NCA 50/90 in the patient's blood is an indication ofcolon cancer in the patient. With this and other information suggestiveof colon cancer, the physician is assisted in making a diagnosis.

The present invention also provides a method for monitoring the courseor progression of colon cancer in a patient who has been diagnosed withcolon cancer. A series of specific immunoassays are performed over timeto determine changes in the level of NCA 50/90 in blood samples obtainedfrom such patient, whereby changes in the NCA 50/90 blood levelcorrelate with changes in disease status. More particularly, increasesin blood NCA 50/90 levels will generally indicate a deterioratingcondition while decreases in blood NCA 50/90 levels indicate animproving condition. Where the diagnosed patient has been treated forcolon cancer, e.g., radiation, chemotherapy, surgery, or the like,increases in blood NCA 50/90 levels indicate recurrence of disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reproduction of a Western blot showing that the 228.2monoclonal antibody (more specifically described hereinbelow, andparticularly in the Examples) binds to NCA 50/90, but does not recognizeNCA 95, CEA, BGPs, or NCA 2. For each of the 3 blots presented, theindicated monoclonal antibodies were blotted against CEA family membersas follows: lane 1, CEA; lane 2, BGP; lane 3, NCA 50/90; lane 4, NCA 95;and lane 5, NCA 2. The control blots in the figure also show that thepreparation of NCA 50/90 used as a standard in the NCA 50/90 ELISA assaycontains only NCA 50/90.

FIG. 2 is a graph showing the standard curve obtained in the NCA 50/90ELISA assay.

FIG. 3 is a graph showing that the NCA 50/90 ELISA assay exhibits nosignificant cross-reactivity with NCA 95, CEA, or BGP.

FIG. 4 is a graph showing that the NCA 50/90 ELISA assay exhibits nosignificant cross-reactivity with α-1-antichymotrypsin, α-1-antitrypsin,α-2-macroglobulin, α-2-anti-plasmin, or antithrombin III.

FIG. 5 is a graph showing that the NCA 50/90 ELISA assay exhibits nosignificant reactivity with serum from pregnant women, demonstrating nosignificant cross-reactivity with PSG.

FIG. 6 is a graph showing that the level of NCA 50/90 is elevated in theserum of patients with colon cancer.

FIGS. 7-12 are a series of graphs showing the course of NCA 50/90 levelsover time in serum samples of individual patients with colon cancer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Essentially any immunological method may be employed in the measurementof blood (e.g., serum or plasma) NCA 50/90 levels. Typically, suchmeasurement will be performed by sandwich immunoassays using twoantibody reagents, one of which recognizes NCA 50/90 to the exclusion ofother related members of the CEA family (e.g., NCA 95, CEA, BGP, PSG,and NCA 2), while the other is capable of binding specifically ornonspecifically with NCA 50/90. Assay format and methods for thepreparation of the required antibody reagents can be selected by theskilled worker in the field. Suitable antibody reagents can be labeled,e.g., enzyme-labeled, or immobilized, e.g., coated onto a microtiterplate, bound to plastic or magnetic beads or particles, and can becomprised of whole immunoglobulins, e.g., IgG or IgM, or fragments,e.g., Fab, Fab′, and F(ab′)₂ fragments, or aggregates thereof.

Preferably, the NCA 50/90 specific antibody reagent is prepared byimmunization of a host animal with a suitable immunogen such as an NCA50/90-containing immunogen mixture, e.g., a purified extract of spleenor tumor cells; NCA 50/90-expressing transfectant cell lines (seeEuropean Patent Publication 346,702); an immunogen conjugate comprisinga synthetically prepared peptide coupled to a conventional immunogeniccarrier molecule, where the peptide has an amino acid sequenceencompassing an epitope of NCA 50/90; and the like as will be understoodin the art.

Antibody reagents comprising monoclonal antibodies will be generallypreferred. Particularly preferred NCA 50/90 specific monoclonalantibodies are those which bind to substantially the same epitope asthat produced by the hybridoma that has deposited on Nov. 18, 1992, withthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852 and which has been given deposit accession number ATCC HB11204. It will be understood that a number of standard methods can beused in order to determine whether a particular monoclonal antibodybinds to substantially the same epitope as the above-mentioned antibodywhose hybridoma has been deposited with the ATCC. A particularly usefulmethod is competitive binding, wherein the ability of the antibody ofinterest to bind to NCA 50/90 in the presence of the reference antibodyis measured. Substantial inability of both antibodies to bindsimultaneously indicates that substantially the same epitope isinvolved.

The present invention also provides an improved method for theimmunoassay determination of the amount of NCA 50/90 in a blood sample,e.g. wherein the blood sample is contacted with an antibody reagent thatis specific for NCA 50/90 and binding between the antibody reagent andNCA 50/90 is determined, preferably by sandwich immunoassay. Theimprovement comprises calibrating the immunoassay by determining bindingof the antibody reagent with a calibrator medium comprisingrecombinantly expressed NCA 50/90.

It will be understood that, similar to other types of accepted diseasediagnostic and monitoring methods, the present method will not be usefulon every patient diagnosed with colon cancer. Rather, the physician willuse NCA 50/90 blood values in combination with other diagnostic valuesand clinical observations to diagnose the onset of colon cancer, andfurther to develop a course of treatment and therapy for each individualpatient. It is also contemplated that monitoring blood levels of NCA50/90 will provide a means for monitoring the progress of a course oftherapy for an individual patient.

The present invention will now be illustrated, but is not intended to belimited by, the following examples.

EXAMPLES

MAb 228.2—BALB/C mice were immunized with 50 μg of an emulsion of NCApurified from human spleen (von Kleist, S. and P. Burtin (1969) CancerRes. 29:1961-1964), and Freund's complete adjuvant. Spleens fromhyperimmune animals were removed from euthanized animals and thesplenocytes were fused with AG8 mouse myeloma cells (ATCC CRL 1580). Theresulting hybridomas were screened for anti-NCA antibody production bysandwich ELISA. Positive clones were subsequently screened for anti-CEAand anti-BGP activity (see Barnett, T. and W. Zimmerman supra; and seeBarnett, T. R., Kretschmer, A., Austen, D. A., Goebel, S. J., Hart, J.T., Elting, J. J., and M. E. Kamarck (1989) J. Cell Biol. 108:267-276)by sandwich ELISA assay. Those clones specific for NCA 50/90 wererecloned and rechecked for cross reactivity with CEA and BGP by ELISAand again by FACS analysis using recombinant mouse cell lines expressingCEA, NCA 50/90 or BGP on their plasma membranes (see European PatentPublication No. 346,702). The result of this screening process wasidentification of MAb 228.2 (deposited with the ATCC, supra) which isspecific for NCA 50/90 with no detectable reactivity with CEA, NCA 95 orBGP by ELISA.

Control MAbs—The 53.5 MAb was derived by procedures similar to thosedescribed above for the 228.2 MAb except that purified CEA from livermetastases (Catalog #CO224, Scripps Laboratories, San Diego, Calif.) wasused as the immunogen and primary screening was by ELISA using purifiedCEA as the antigen. This antibody reacts on Western blots with CEA, BGP,NCA 50190, NCA 95 and NCA 2, and is used as a positive control.

The MAb designated 030-A1101 is an IgG1 which binds to alpha fetoprotein(AFP), and was obtained from BiosPacific (Emeryville, Calif.). It wasused in these studies as a negative control.

Biotinylation of Goat Antibody to CEA—An affinity purified polyclonalgoat antiserum raised to CEA was purchased from BiosPacific, Emeryville,Calif., USA (Lot No. 015-B4302) and placed into 1.3 ml of 0.1M NaHCO₃,pH 8.5 at a concentration of 1.0 mg/ml. To this was added 18.5 μl of a10 mg/ml solution of NHS-LC-biotin (Pierce, Rockford, Ill., USA, CatalogNo. 21335) in deionized water to give a 50/1 molar excess of biotin toantibody. After incubation at 0° C. for 4 hours the biotinylatedantibody was passed over a buffer-exchange column using 10 mM phosphate,pH 7.4/150 mM NaCl and stored at 4° C. with 0.1% thimerosal aspreservative.

NCA Calibrator—A cDNA corresponding to NCA 50/90 was derived from thebreast tumor cell line BT-20 as described previously (Barnett, T.,Goebel, S. J., Nothdurft, M. A. and J. J. Elting (1988) Genomics 3,59-66). The coding region for the NCA 50/90 gene was modified by theelimination of the C-terminal hydrophobic region which signalsreplacement by a phosphoinositol glycan linkage, and the addition of astretch of six histidine residues, also at the carboxyl terminus of themolecule (Drake, L. and Barnett, T. (1992) Biotechniques 12, 645-649).This construct was cloned into pVL1393 by PCR and expressed usingrecombinant baculovirus to infect Spodoptera frugiperda (Sf9) cells. NCA50/90 was affinity purified from Sf9 supernatant fluids using azinc-imidoacetate-Sepharose® column as described (Drake and Barnett,supra). The concentration of NCA was determined by the BCA protein assay(Pierce, Cat. No. 23225G). For use as a calibrator in the NCA 50/90ELISA, purified recombinant NCA 50/90 was diluted in TBST/5% BSA asdescribed below.

NCA 50/90-Specific Immunoassay—A sandwich ELISA was configured using the228.2 monoclonal antibody as the solid phase capture antibody, and thebiotinylated polyclonal anti-CEA as the reporter antibody. 96-well BLISAplates (Immulon 4, Dynatech Laboratories, Chantilly, Va., USA) werecoated with 100 μl of 228.2 antibody at 5 μg/ml in 0.1M NaHCO₃, pH 9.5and incubated overnight at 4° C. Wells were emptied and unreacted siteson the plates were quenched by the addition of 200 μl of 20 mM Tris, pH7.5/150 mM NaCl/0.05% Tween 20 (TBST) with 5% bovine albumin (BSA,fraction V, Sigma Chemical Company, St. Louis, Mo., USA, Catalog No.A-7030) followed by a 1 hour incubation at 37° C. Wells were washed 6times with TBST, and 25 μl of either NCA 50/90 calibrators diluted inTBST/5% BSA or 25 μl of patient sample was added. An equal volume of 50mM HEPES, pH 7.0/500 mM NaCl/200 μg/ml mouse IgG/5% BSA/50 μg/mlgentamycin/0.1% (w/v) NaN₃ (sample diluent) was added to each well andthe plates were incubated for 2 hours at 37° C. After washing 6 times, a100 μl volume of a 0.3 μg/ml solution of goat anti-CEA-biotin in 50 mMHEPES, pH 7.0/150 mM NaCl/1 mM MgCl₂.6H₂O/0.1 mM ZnCl₂/5% BSA/50 μg/mlgentamycin/0.1% NaN₃ (conjugate diluent) was added to all wells andincubated for 1 hour at 37° C. The wells were washed a further 6 times,and 100 μl of streptavidin conjugated to alkaline phosphatase (Pierce,Catalog No. 21324G) diluted 1/5000 in conjugate diluent was added. Aftera 1 hour incubation at 37° C., the plates were washed 12 times with TBSTand incubated with 100 μl of p-nitrophenyl phosphate at 1 mg/ml indiethanolamine substrate buffer (Pierce, Catalog No. 34064) for 30minutes. The reaction was stopped with 100 μl 1N NaOH and absorbance at405 nm minus absorbance at 490 nm determined using a microplate reader(ThermoMax, Molecular Devices Corp., Menlo Park, Calif., USA). Theamount of NCA 50/90 was determined for each test sample by comparisonwith the calibrator standard curve.

Patient Samples—Serum was prepared from blood drawn from normal healthyvolunteers by Hudson Valley Blood Services of Valhalla, N.Y., USA.Plasma samples from patients with inactive or active colon cancer wereobtained from Dianon Systems of Stratford, Conn., USA. Longitudinalserum samples drawn from individual patients during their period oftreatment were obtained from M.D. Anderson Cancer Center in Houston,Tex., USA. Patient disease status was determined from informationsupplied by attending physicians as well as results of testing for thetumor markers CEA, lipid associated sialic acid (LASA), and CA 19-9.

Results

The antigenic specificity of the 228.2 monoclonal antibody wasdetermined first by Western blotting and results are shown in FIG. 1.The 228.2 MAb reacts specifically with NCA 50/90 and not with otherproteins related to CEA. The reactivity of the 228.2 MAb with the highmolecular weight band of M_(r) 110,000 probably represents the formationof SDS-stable protein dimers. The reactivity of the 228.2 MAb with theNCA 2 preparation is with an M_(r) 90,000 protein which does notcomigrate with the M_(r) 160,000 NCA 2 protein, and is likely torepresent a low level of contamination of the NCA 2 preparation with theM_(r) 90,000 form of NCA 50/90. The reactivity of the positive controlMAb 53.5 with each of the antigen preparations demonstrates the presenceof the relevant glycoproteins in each preparation. In addition, the 53.5MAb reacted only with a protein of M_(r) 50,000 in the NCA 50/90preparation, which demonstrates the antigenic purity of the NCA 50/90preparation. The MAb 030-A11101 binds to alpha fetoprotein and was usedas negative control.

The standard curve presented in FIG. 2 demonstrates a nonlinear increasein absorbance as a function of NCA 50/90 concentration. A nonlinearspline curve fit program was used to convert raw patient data to NCA50/90 concentrations. The data in FIGS. 3 and 4 demonstrate that the NCA50/90 ELISA shows no significant reactivity with CEA, NCA 95, BGPs,α-1-antichymotrypsin, α-1-antitrypsin, α-2-macroglobulin,α-2-antiplasmin and antithrombin III. The potential for cross reactivitywith serine proteinase inhibitors stems from observations thatbiochemically purified CEA and NCA may associate with molecules withamino acid homology to α-1-antichymotrypsin and α-1-antitrypsin(Orjaseter, H. (1976) Acta Path. Microbiol. Scand. 84, 235-244; andGrunert, F., Abuharfeil, N., Luckenbach, G. A. and S. von Kleist (1984)Tumor Biol. 5, 221-232). Since the MAb 228.2 was raised to biochemicallypurified NCA 50/90 from spleen, there is some potential for crossreactivity with contaminating proteins.

An additional member of the CEA gene family is pregnancy specific6-glycoprotein (PSG) which is elevated in the serum of pregnant women.Reactivity to this protein was tested by examining sera from 15 pregnantwomen with HCG values ranging from 2200 to 79,000 (normal cutoff forHCG=10). As can be seen in FIG. 5, only one patient showed an NCA valueabove the cutoff value of 18 ng/ml, which demonstrates that the NCA50/90 ELISA does not detect PSG.

To establish a cutoff value for normal blood levels of NCA 50/90, thelevel of NCA 50/90 in serum was measured from 92 normal healthyvolunteers. A 95% cutoff value was determined to be 18 ng/ml. NCA 50/90values in plasma from 10 colon cancer patients undergoing treatment whowere clinically free of cancer was then measured, and it was found that100% of the values were below the cutoff value. In contrast, 31 of 62samples from patients with active colon cancer were above the cutoffvalue, which demonstrates that NCA 50/90 is elevated above normal levelsin the blood of some patients with colon cancer. The high incidence ofelevated values of NCA 50/90 in the blood of colon cancer patients showsthat the present method is useful to detect cancer in patients.

The serum level of NCA 50/90 was found to correlate with the status ofdisease in 20 patients diagnosed with and under treatment for coloncancer. Representative results obtained with six such patients arepresented in FIGS. 7-12.

The terms used in FIGS. 7-12 to denote patient status are defined asfollows:

NED—No clinical evidence of disease as determined by the attendingphysician and verified by normal blood levels of at least twobiomarkers.

PRO—Clinical evidence of progressive disease as determined by theattending physician and verified by elevated blood levels of at leastone cancer biomarker.

STAB—Clinically stable cancer with no evidence of disease progressionsince the last examination.

RESP—Responding to treatment with at least a 50% decrease in the tumormass since diagnosis.

Patient CS8 was disease free throughout the course of the study, andboth CEA and NCA 50/90 levels were below cutoff values. Patients CS12,CS15 and CS20 had active disease at the time points that samples weretested for NCA 50/90, and NCA 50/90 values were elevated above cutoff inall cases. Results with patient CS19 show a clear increase in NCA 50/90values which preceded clinical evidence of recurrence. CEA values,however, were not elevated prior to clinical evidence of recurrentdisease. In contrast, results with patient CS17 show that while CEA waselevated when disease recurred in month 7 of the study, the NCA 50/90values were not elevated. The combined results with all 20 patientsdemonstrate that NCA 50/90 values correctly reflected disease status in88% of the longitudinal samples. Taken together, these resultsdemonstrate that NCA 50/90 can be used to monitor disease status incolon cancer patients under treatment. In addition, NCA 50/90 iselevated in a different population of patients that currently usedbiomarkers, such as CEA.

The present invention has been particularly described and exemplifiedabove. Clearly, many other variations and modifications of the inventioncan be made without departing from the spirit and scope hereof.

What is claimed is:
 1. A method for monitoring the course of disease ina patient diagnosed with colon cancer, comprising the performance of aseries of specific immunoassays over time to determine changes in thelevel of NCA 50/90 in blood samples obtained from such patient, wherebychanges in the NCA 50/90 blood level correlate with changes in diseasestatus.
 2. The method of claim 1 wherein the immunoassays performed aresandwich immunoassays in which at least one of the antibody reagents isspecific for NCA 50/90 with no substantial reactivity for CEA, NCA 95,or BGP.
 3. The method of claim 2 wherein said NCA 50/90 specificantibody reagent is a monoclonal antibody reagent.
 4. The method ofclaim 2 wherein said NCA 50/90 specific antibody reagent binds to thesame epitope as the monoclonal antibody produced by the hybridomadeposited by the present inventors with the American Type CultureCollection (ATCC), Rockville, Md., USA, and identified as ATCC HB 11204.5. The method of claim 1 wherein said blood samples are serum or plasmasamples.
 6. A method for monitoring the course of disease in a patientdiagnosed with colon cancer, comprising the performance of a series ofspecific immunoassays over time to determine changes in the level of NCA50/90 in blood samples obtained from such patient, whereby increases inblood NCA 50/90 levels indicate a deteriorating condition Whiledecreases in blood NCA 50/90 levels indicate an improving condition. 7.The method of claim 6 wherein the immunoassays performed are sandwichimmunoassays in which at least one of the antibody reagents is specificfor NCA 50/90 with no substantial reactivity for CEA, NCA 95, or BGP. 8.The method of claim 7 wherein said NCA 50/90 specific antibody reagentis a monoclonal antibody reagent.
 9. The method of claim 7 wherein saidNCA 50/90 specific antibody reagent binds to the same epitope as themonoclonal antibody produced by the hybridoma deposited by the presentinventors with the American Type Culture Collection (ATCC), Rockville,Md., USA, and identified as ATCC HB
 11204. 10. The method of claim 6wherein said blood samples are serum or plasma samples.
 11. A method formonitoring the course of disease in a patient who has been treated forcolon cancer, comprising the performance of a series of specificimmunoassays over time to determine changes in the level of NCA 50/90 inblood samples obtained- from such patient, whereby increases in bloodNCA 50/90 levels indicate recurrence of disease.
 12. The method of claim11 wherein the immunoassays performed are sandwich immunoassays in whichat least one of the antibody reagents is specific for NCA 50/90 with nosubstantial reactivity/for CEA, NCA 95, or BGP.
 13. The method of claim12 wherein said NCA 50/90 specific antibody reagent is a monoclonalantibody reagent.
 14. The method of claim 12 wherein said NCA 50/90specific antibody reagent binds to the same epitope as the monoclonalantibody produced by the hybridoma deposited by the present inventorswith the American Type Culture Collection (ATCC), Rockville, Md., USA,and identified as ATCC HB
 11204. 15. The method of claim 11 wherein saidblood samples are serum or plasma samples.